花椒窄吉丁气味结合蛋白AzanOBP5的克隆与表达  

Cloning and expression of the odorant binding protein AzanOBP5 in pepper buprestid beetle Agrilus zanthoxylumi

作　　者：郭莉 陈迪 贾任航 高晓进 张泽腾 谢寿安[1] 吕淑杰[1] Guo Li;Chen Di;Jia Renhang;Gao Xiaojin;Zhang Zeteng;Xie Shou’an;LüShujie(College of Forestry,Northwest A&F University,Yangling 712100,Shaanxi Province,China)

机构地区：[1]西北农林科技大学林学院,陕西杨凌712100

出　　处：《植物保护学报》2023年第1期81-90,共10页Journal of Plant Protection

基　　金：国家公益性行业(林业)科研项目(201504324);云南省林业科技推广示范项目(云[2019]TG01号);陕西省第二批特支计划领军人才项目(陕组通字2020-44号);2022年度西安市科技计划项目(2022JH-JSYF-0261)。

摘　　要：为进一步明确花椒窄吉丁Agrilus zanthoxylumi气味结合蛋白AzanOBP5在昆虫嗅觉识别过程中的功能,基于花椒窄吉丁转录组数据(SUB6796283)利用cDNA末端快速扩增技术对花椒窄吉丁气味结合蛋白基因AzanOBP5进行全长克隆并进行生物信息学分析;通过实时荧光定量检测技术测定基因AzanOBP5在成虫不同组织中的表达情况。结果显示,克隆获得基因AzanOBP5全长740 bp(GenBank登录号为MT318834),5’端非编码区为172 bp,3’端非编码区114 bp,开放阅读框453 bp,编码150个氨基酸。序列分析表明,AzanOBP5氨基酸序列与苹果小吉丁Agrilus mali AmalOBP5的氨基酸序列一致性最高。成功构建pET-21a-AzanOBP5重组质粒并在大肠杆菌Escherichia coli中表达,经过诱导和纯化条件的优化及Western blot鉴定,得到的融合蛋白His-AzanOBP5大小符合预期。基因AzanOBP5在雌雄成虫的不同组织中均有表达,其中在成虫腹部的表达量最高。表明花椒窄吉丁气味结合蛋白基因AzanOBP5除参与嗅觉识别过程外,还可能具有其他生理功能。To clarify the function of AzanOBP5,an odorant binding protein in pepper buprestid beetle Agrilus zanthoxylumi,in insect olfactory recognition.RACE technology was used to clone the fulllength cDNA of AzanOBP5 based on the transcriptome data of A.zanthoxylumi(SUB6796283).The nucleotide and deduced amino acid sequences were analyzed by bioinformatics software.The expression levels of AzanOBP5 in different tissues of adult were determined by real time quantitative PCR.The full-length cDNA of AzanOBP5 was 740 bp(GenBank accession no.:MT318834),with a non-coding region of 172 bp and 114 bp at the 5′and 3′ends,respectively.The open reading frame was 453 bp,encoding 150 amino acids.Homologous sequence alignment analyses showed that AzanOBP5 had the highest amino acid sequence identity with AmalOBP5 from A.mali.The recombinant plasmid pET-21aAzanOBP5 was successfully constructed and expressed in E.coli.After optimization of the induction and purification conditions,it was identified by Western blot.The obtained fusion protein His-AzanOBP5 was in line with expectations.The gene AzanOBP5 was expressed in various tissues of both male and female adults,and the highest expression level was detected in abdomen.The study showed that AzanOBP5 not only played a role in olfactory recognition,but also had other physiological functions in A.zanthoxylumi.

关 键 词：花椒窄吉丁 气味结合蛋白 基因克隆 组织表达 原核表达 

分 类 号：S435.73[农业科学—农业昆虫与害虫防治]